Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1

Leoh, Lai Sum; Daniels‐Wells, Tracy R.; Martı́nez-Maza, Otoniel; Penichet, Manuel L.

doi:10.1016/j.molimm.2015.07.001

Cited by 16 publications

(24 citation statements)

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“…Our recombinant antibody (IgG3KVH) showed stronger binding to C1q than IgG1. Previously, it was reported that residues D270, K322, P329, and P331 in the CH2 domain constitute the human C1q binding site of the Fc region . However, the substitutions introduced into IgG3KVH were all within the CH3 region.…”

Section: Discussionmentioning

confidence: 99%

“…For the IgG3KV antibody, FcγRIIIa binding activity was drastically decreased. It has also been shown that IgG1 binding to FcγRIIIa is mediated through the hinge and CH2 regions and that the CH3 domain does not directly affect binding . A previous study indicated that the CH3 and CH2 domains of the IgG2 antibody interacted via L251 in the CH2 region and residues M428, H429, E430, and H435 in the CH3 domain .…”

Section: Discussionmentioning

confidence: 99%

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A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

et al. 2019

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Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti‐CD20 IgG3 as a model to investigate aggregate formation. Initially, anti‐CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild‐type IgG3. IgG3KVH also exhibited high binding activity for FcγRIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

et al. 2019

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“…ch128.1 (IgG3/κ) and the ch128.1 triple mutant L234A/L235A/P329S were produced in murine myeloma cells and affinity purified as described (27, 28). Mutations were previously generated on the ch128.1 heavy chain γ3 expression vector to disrupt binding to FcγRs and complement component C1q (27).…”

Section: Methodsmentioning

confidence: 99%

Efficacy and Mechanism of Antitumor Activity of an Antibody Targeting Transferrin Receptor 1 in Mouse Models of Human Multiple Myeloma

Leoh

Kim

Candelaria

et al. 2018

The Journal of Immunology

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The transferrin receptor 1 (TfR1) is an attractive target for Ab-mediated cancer therapy. We previously developed a mouse/human chimeric IgG3 Ab (ch128.1) targeting human TfR1, which exhibits direct in vitro cytotoxicity against certain human malignant B cells through TfR1 degradation and iron deprivation. ch128.1 also demonstrates exceptional antitumor activity against the B cell malignancy multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing either disseminated ARH-77 or KMS-11 cells in an early disease setting. Interestingly, this activity is observed even against KMS-11 cells, which show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. To understand the contributions of the Fc fragment, we generated a ch128.1 mutant with impaired binding to FcγRs and to the complement component C1q, which retains binding to the neonatal Fc receptor. We now report that this mutant Ab does not show antitumor activity in these two MM models, indicating a crucial role of the Fc fragment in the antitumor activity of ch128.1, which can be attributed to effector functions (Ab-dependent cell-mediated cytotoxicity, Ab-dependent cell-mediated phagocytosis, and/or complement-dependent cytotoxicity). Interestingly, in the KMS-11 model, complement depletion does not affect protection, whereas macrophage depletion does. Consistent with this observation, we found that ch128.1 induces Ab-dependent cell-mediated cytotoxicity and Ab-dependent cell-mediated phagocytosis against KMS-11 cells in the presence of murine bone marrow-derived macrophages. Finally, we found that ch128.1 therapy effectively increases survival in a late MM disease setting. Our results suggest that macrophages play a major role in ch128.1-mediated antitumor protection in our models and that ch128.1 can be effective against human B cell malignancies such as MM.

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“…Another example is an Fc-engineered anti-CD70 IgG1 that contains five mutations (C226S/C229S/E233P/L234V/L235A) and shows decreased ADCP activity ( 123 ). Furthermore, a human IgG3 targeting the transferrin receptor 1 (TfR1) containing only two mutations (L234A/L235A) showed decreased ADCC activity against TfR1 expressing target cells, an effect that was increased by the addition of a third mutation P329S ( 125 ). However, the P329S single mutant showed no effect on ADCC ( 125 ).…”

Section: Modulating Fc-dependent Effector Functionsmentioning

confidence: 99%

“…Point mutations at any one of these sites in rituximab decreased CDC activity, but not ADCC or binding to FcRn ( 129 ). In a human IgG3 targeting TfR1, the P329S mutation abolished CDC activity against TfR1 expressing target cells, but no impaired ADCC was detected with this single mutation ( 125 ).…”

Section: Modulating Fc-dependent Effector Functionsmentioning

confidence: 99%

Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

Almagro¹,

et al. 2018

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The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA) of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life), which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

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Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1

Cited by 16 publications

References 50 publications

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Efficacy and Mechanism of Antitumor Activity of an Antibody Targeting Transferrin Receptor 1 in Mouse Models of Human Multiple Myeloma

Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy

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